The 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl] quinazoline salts ("trimetrexatel") are known to have antineoplastic, antiparasitic and antibacterial activity. Trimetrexate is a potent inhibitor of the enzyme dihydrofolate reductase ("DHFR") that is known to catalyze the synthesis of nucleotide precursors of DNA and RNA. Inhibition of this enzyme results in cellular depletion of reduced folates, which are necessary for various cellular process, including RNA and DNA synthesis, and ultimately results in cell death. It is this property that gives trimetrexate its antineoplastic, antiparasitic and antibacterial activity.
Trimetrexate has demonstrated antitumor activity against a range of experimental murine and human tumor cell lines, in vitro and in vivo. For example, trimetrexate has shown antitumor activity against murine cell lines such as L1210, L5178Y, S-180, W-256 in vitro. In addition, trimetrexate has shown antitumor activity against human tumor cells lines derived from breast, colon, lung, ovary, renal cell and melanoma cells in vitro. In vivo studies have demonstrated that trimetrexate has therapeutic utility against murine tumors such as B16 melanoma, colon 26 and 38, L1210 and p388 leukemia and CD8F mammary tumors. Other possible uses for trimetrexate include the treatment of malaria, psoriasis, rheumatoid arthritis and prophylaxis against Pneumocystis carinii pneumonia.
Trimetrexate as the free base is a lipophilic DHFR with very low water solubility. To increase the aqueous solubility the Warner-Lambert Company synthesized a number of various salts which were disclosed by Colbry in U.S. Pat. No. 4,376,858 and issued on Mar. 15, 1983 and assigned to the Warner-Lambert Company. The preferred salt reported by Colbry was trimetrexate glucuronate due to its superior water solubility, stability and low toxicity of glucuronic acid. The method disclosed by Colbry gives a method of preparation where trimetrexate and glucuronic acid are dissolved in warm methanol and ethyl acetate and followed by cooling to precipitate the salt. The Colbry patent states that the disclosed trimetrexate glucuronate salt is non-crystalline.
J. L. Hicks et al., J. Labelled Compound Radiopharm. 29, 415 (1991) disclose another method of manufacture of trimetrexate glucuronate salt. In this method trimetrexate free base and glucuronic acid are added to an aqueous solution in an ampoule, followed by lyophilization to form a solid. This method also produces an amorphous salt.
An injectable form of amorphous trimetrexate glucuronate has received marketing authorization in the United States and Canada under the trade name Neutrexin by U.S. Bioscience (West Conshohocken, Pa.) for use in combination with leucovorin for the treatment of moderate to severe Pneumocystis carinii pneumonia in immunocompromised patients, e.g., those suffering from AIDS (U.S. Bioscience's Neutrexin for PCP, Scripp 1886/87, 31(1994).
A. Hempel et al., Cancer Biochem. Biophys. 10, 25-30 (1988), discloses the crystal structures of trimetrexate acetate monohydrate and hydrated trimetrexate free base. There is no suggestion regarding crystalline trimetrexate glucurotiate or the method of making the same.
H. K. Chan et al., J. Crystal Growth 94, 488-498 (1989) discloses techniques for modifying the crystallization of methotrexate. The presence of trimetrexate in solution was shown to affect the crystalline habit of the methotrexate that is precipitated. There is no suggestion regarding the crystallization of trimetrexate glucuronate.
P. L. Stetson et al., J. Chromatography 464, 163-71 (1989) discusses the stability of the trimetrexate glucuronate salt: obtained from Warner Lambert/Parke-Davis, Pharmaceutical Research Division. This salt is apparently that obtained by the process disclosed in the U.S. Pat. No. 4,376,858, which is assigned to Warner Lambert. It indicates that the salt has a half-life in solution of 51.6.+-.0.8 days at 37.degree. C.
It is recognized that the amorphous form of trimetrexate salts is not the preferred form as a drug substance for oral administration. Accordingly, there is a strong need in the art for suitable methods for producing quantities of highly purified, medical grade trimetrexate salts having a crystalline structure, in order to obtain high and uniform stability during storage, and to provide oral forms of trimetrexate medications with good stability and bioavailability.